Project Summary BACKGROUND: Glioblastoma is the most common form of primary adult brain cancer and remains a deadly disease. The standard of care for glioblastoma involves surgical resection followed by radiation and temozolomide chemotherapy (TMZ). While Progression Free Survival (PFS) studies have revealed that nearly 80% of treated patients are responsive to TMZ at 6 months, only approximately 10% of these patients remain responsive by 24 months. We investigated whether altered microRNA (miRNA) expression during TMZ treatment contributes to acquired TMZ resistance. To this end, we profiled miRNAs in matched pre- and post- TMZ treated glioblastoma cell lines and clinical specimens. We identified one miRNA, miR-181d, which is down-regulated in response to TMZ treatment in both of these settings and profoundly influenced cellular TMZ sensitivity. miR-181d normally suppresses the expression of multiple DNA repair genes critical for TMZ resistance, including Methyl-Guanine Methyl-Transferase (MGMT) and homologous recombination (HR) genes. TMZ-induced miR-181d degradation up-regulates both processes and contributes to TMZ resistance. This proposal will characterize miRNA degradation as an acquired resistance mechanism and develop a therapy that targets this resistance. AIM 1 proposes experiments to characterize a) the molecular mechanisms by which the DNA damage response triggers miRNA degradation and b) the relevance of this process to acquired TMZ resistance. AIM 2 proposes experiments to a) characterize the genetic context in which miR- 181d degradation contributes to acquired TMZ resistance and b) determine the miR-181d regulated processes that contribute to this resistance. AIM 3 is built on the premise that over-expression of miR-181d beyond cellular capacity for degradation will suppress acquired TMZ resistance; to that end, we propose a miRNA based gene therapeutic approach to address the issue of acquired TMZ resistance. INNOVATION: The proposed study to characterize TMZ-induced miRNA degradation as a novel mechanism of acquired TMZ resistance is an innovative and heretofore unexplored approach. Moreover, this proposal develops an original framework that miRNA degradation simultaneously up-regulates multiple DNA repair processes that, in turn, contribute to TMZ resistance. Finally, we propose an innovative therapeutic strategy for addressing this form of resistance. LONG-TERM OBJECTIVE: We seek to meaningfully impact the care of glioblastoma patients through the application of principles developed in the fields of DNA repair, microRNAs, and retroviral gene therapy.